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SNP markers for identification of low stone cell content in pear pulp based on high-resolution melting curves and their application

A high-resolution, melting curve technology, applied in the field of SNP markers, can solve the problems that the development and application of SNP molecular markers have not yet been reported.

Active Publication Date: 2021-01-12
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the research on QTL mapping of quantitative traits in pears is still in its infancy. The existing researches are mainly aimed at some diseases or growth traits. There is no report on the development and application of SNP molecular markers for low stone cell content in pear pulp.

Method used

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  • SNP markers for identification of low stone cell content in pear pulp based on high-resolution melting curves and their application
  • SNP markers for identification of low stone cell content in pear pulp based on high-resolution melting curves and their application
  • SNP markers for identification of low stone cell content in pear pulp based on high-resolution melting curves and their application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] The mensuration of embodiment 1 stone cell content

[0025] Stone cells were separated and weighed according to the method of Tao et al. (2009) [6]. Peel and remove the core of the fruit, take 100g of pulp and add distilled water to beat, then dilute the homogenate with distilled water, stir and let it stand at room temperature for 30 minutes, discard the floating matter, resuspend the sediment in 0.5mol / L hydrochloric acid for 30 minutes, discard Floats were washed with distilled water. Repeat the above steps until the stone cells are separated from other cell fragments, and weigh after drying. Table 1 shows the content of stone cells in the flesh of 56 pear natural populations at 35 days after flowering.

[0026] Table 1 Stone cell content in the pulp of 56 pear natural populations at 35 days after flowering

[0027]

[0028]

[0029] Note: Serial numbers 1-30 are varieties with high stone cell content, and 31-56 are varieties with low stone cell content.

Embodiment 2

[0030] Example 2 Development of HRM-specific primers using SNP marker sites Psc397-7 and Psc397-12

[0031] The DNA sequences of SNP markers Psc397-7 and Psc397-12 on the fifth chromosome were searched from the whole genome database of Dangshan Suli, and the 300 bp sequences before and after the site were selected, and the primers for SNP markers were developed and designed according to the principle of primer design (Table 2). Using the designed SNP marker primers to carry out PCR amplification on the genomic DNA of Dangshansu pear, the primers were amplified normally, and the PCR product was in line with the predicted size.

[0032] Table 2 SNP marker primers

[0033]

Embodiment 3

[0034]Example 3 Using HRM (high-resolution melting, high-resolution melting curve) technology to type pear pulp stone cell content

[0035] The HRM reaction system was performed according to the instructions in the LightCycler 480 High Resolution Melting Master kit, and the HRM analysis was performed on a LightCycler480 II fluorescent quantitative PCR instrument. 20μL reaction system: containing 30ng pear genomic DNA template, 1×Master Mix, 2.5mmol / L MgCl 2 , 200 nM of the primers described (Psc397-7-F: SEQ ID NO.1 / Psc397-7-R: SEQ ID NO.2 or Psc397-12-F: SEQ ID NO.3, / Psc397-12-R: SEQ ID NO.4); the amplification program adopts touchdown PCR (touchdown PCR): pre-denaturation at 95°C for 10 minutes, then denaturation at 95°C for 10 seconds, annealing at 65-55°C (0.5°C per cycle) for 10 seconds, and extension at 72°C for 10 seconds The program was performed for 45 cycles. Melting was performed after the PCR cycle was completed. The program was: 95°C for 1 min, 40°C for 1 min, 6...

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Abstract

The invention discloses a SNP molecular marker primer closely related to the pear pulp low stone cell content. The SNP markers closely related to the pear pulp low stone cell content are Psc397-7, Psc397-12, and the primers are SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4. Based on a high resolution dissolution curve, the specific primers can well type the pulp low stone cell content. The pear pulp stone cell content molecular marker primer can be used in molecular marker assisted selection breeding of the pear pulp low stone cell content, and has great theoretical and practical guiding significance to speed up the genetic improvement process of pear varieties and improve the selection efficiency of breeding.

Description

technical field [0001] The invention belongs to the field of molecular genetic breeding, and relates to a SNP marker for identifying low stone cell content in pear pulp based on a high-resolution melting curve and an application thereof. Background technique [0002] Pear has a long history of cultivation in my country, and it is also an important deciduous fruit tree in the world. Pear fruit has high nutritional value, crisp and juicy meat, and is deeply loved by people. The edible quality of pear fruit has always been a concern of people, and it is an important factor determining the economic value of pear fruit, so it is of great significance to improve the edible quality of pear fruit. The eating quality of pear fruit is affected by many factors, among which stone cells are one of the important factors affecting the quality of pear fruit. Stone cells are a special type of thick-walled cells in pear fruit, which not only affect the eating quality of pear fruit, but also...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6895C12Q2600/13C12Q2600/156C12Q2527/107C12Q2563/107
Inventor 吴俊薛程张绍铃谷超张明月秦梦帆殷豪谢智华
Owner NANJING AGRICULTURAL UNIVERSITY
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